The wide variety of microorganisms commonly found in the gastrointestinal tract, particularly the Gram-negative, nonsporulating bacilli, have become increasingly important in clinical medicine. They are the principal organisms found in infections of the abdominal viscera, peritoneum, and urinary tract, as well as being frequent secondary invaders of the respiratory tract, burned or traumatized skin, and sites of decreased host resistance and instrumentation. Currently, they are the most frequent cause of life-threatening bacteremia.
The gastrointestinal flora are exceedingly complex. The large intestine contains about 10.sup.10 to 10.sup.11 organisms per gram of contents. Of these, 90 to 95 percent are obligate anaerobes. Most common are the Gram-negative bacilli, Bacteroides and Fusobacterium, gram-positive bacilli, including Bifidobacterium, Eubacterium, and Corynebacterium species, and a wide variety of anaerobic streptococci. Other anaerobes include the Gram-positive spore-forming rods of the clostridia species and Gram-negative cocci, Veillonella.
Enterococci are also present. The well-known aerobic Gram-negative rods, which are members of the family Enterobacteriaceae, account for only 5 to 10 percent of the total flora. These include the most common, E. coli , as well as the Klebsiella-Enterobacter group, Proteus, Providencia, Edwardsiella, Serratia, and under pathologic conditions, Salmonella and Shigella.
The Gram-negative bacteria of the gastrointestinal tract produce disease by invasion of tissue and by release of a pharmacologically active lipopolysaccharide (LPS) from the cell wall, known as endotoxin. Endotoxins from a wide variety of unrelated species behave quite similarly, regardless of the inherent pathogenicity of the microorganism from which they are derived or their antigenic structure.
In the intact microorganism, endotoxins exist as complexes of lipid, polysaccharide, glycolipid and non-covalently-bound protein. The biologic activity seems to be a property of a lipid and carbohydrate portion.
The lipopolysaccharides of Gram-negative bacteria may be roughly divided into three structural regions. The outer-most region contains the chains of specific sugars that characterize the O-specific antigens and determine individual serotypes within a species. The specific sugars are linked to a core polysaccharide that is of similar structure among related groups of bacteria. The core is in turn linked through 2-keto-3-deoxyoctonate disaccharides to the major lipid component termed lipid A. Evidence has now accumulated to indicate that the properties of endotoxins may be accounted for by this complex lipid substance.
Lipid A is a glucosamine disaccharide esterified with phosphoric and pyrophosphoric acid and also contains ester- or amide-linked lauric, palmitic, and myristic acids. Perhaps the most important finding in recent years is that the lipid A and core-polysaccharide regions are immunogenic and can induce antibodies that cross-react among the Gram-negative bacteria.
Animal studies reveal that antibodies prepared against these components of endotoxin protect against challenge from heterologous Gram-negative bacteria. However, better protection is reportedly obtained by immunization with specific O-antigens that induce opsonizing antibodies.
Upon entry into the bloodstream and initiation of endotoxemia, LPS and blood humoral and cellular elements interact. The work of several groups has shown that the blood-borne LPS partitions between the tissues and plasma lipoproteins, with specific binding to high density lipoprotein (HDL). Freudenberg et al. (1980) Infec. Immun. 28:373; Mathison and Ulevitch (1979) J. Immunol. 123:2133; Munford et al. (1982) J. Clin. Invest. 70:877; and Ulevitch et al. (1981) J. Clin. Invest. 67:827.
Depending upon the source and isolated form of LPS, about 10-50 percent of the initially administered LPS partitions to the plasma lipoproteins (HDL), with the remainder going to the tissues. Clearance from the animal body appears to be via the tissues and into bile. Thus, if the partitioning between plasma lipoproteins and tissues could be adjusted to be less favorable to HDL, LPS could be more quickly cleared from the body of the infected animal.
In view of the above reports and the findings discussed hereinafter, an early example of improved clearance may have been reported by Filkins (1976) Proc. Soc. Exptl. Biol. Med. 151:89. It was there reported that rats treated with whole rat blood plasma and serum from either post-endotoxic or post-trauma donors manifested detoxifying potential in rats into which Salmonella enteritidis LPS had been injected. In contrast, normal rat blood and phosphate-buffered saline controls exhibited no detoxification. A role for the reticuloendothelial system in elaboration of the blood anti-endotoxin system was postulated.
It has also been reported that acute phase rabbit serum (APRS) modifies the interactions of LPS with HDL by retarding the in vitro rate of binding of LPS to HDL, thereby modulating the endotoxic effect of the LPS. Tobias and Ulevitch (1983) J. Immunol. 131:1913. Binding of LPS to components of normal rabbit serum (NRS) has also been reported. Ulevitch and Johnston (1978) J. Clin. Invest. 62:1313; Ulevitch et al. (1979) J. Clin. Invest. 64:1516; Ulevitch et al. (1981) J. Clin. Invest. 67:827; Munford et al. (1981) J. Clin. Invest 70:877; and Freudenberg et al. (1980) Infect. Immun. 28:373.
In rabbits, the interaction of LPS with HDL can be accounted for by a two-step mechanism in which the LPS is first disaggregated by the action of serum proteins. The disaggregated LPS thereafter binds with HDL to form the observed complex. It is believed that similar mechanisms apply in other animals, including man.
Mixtures of LPS with rabbit serum that are permitted to react for 30 minutes at 37 degrees C provide an LPS complex with a density of less than about 1.2 grams per cubic centimeter (g/cc). When NRS is used, the complex contains components of HDL including apolipoprotein AI (apo AI) [Ulevitch et al. (1981) J. Clin. Invest. 67:827], while with APRS the complex contains apo AI and also serum amyloid A apolipoprotein (apo SSA) [Tobias et al. (1982) J. Immunol. 128:1420].
While complexes with densities of about 1.2 g/cc are ultimately formed by admixture of LPS with NRS and with APRS, the times for formation of similar amounts of those complexes differ. Thus, for NRS, the formation of the 1.2 g/cc complex is about 90 percent complete within about 30 minutes, while in APRS, the complex is about 95 percent formed after a time period of about 6 hours. Tobias and Ulevitch (1983) J. Immunol. 131:1913.
In addition to the time courses of complex formation being different in NRS as compared to APRS, initial complexes formed in the two serum types also differ in density. Thus, in NRS, the density of the initially formed complex is 1.33 g/cc, while in APRS, the density of the intial complex is 1.3. An LPS-containing serum complex with a density of 1.3 g/cc was also reported when Balbc/Strong mice were injected with AgNO.sub.3 or LPS. Tobias and Ulevitch (1983) J. Immunol. 131:1913.
Precipitated euglobulin fractions formed from mixtures of LPS and NRS or APRS were examined for their solubilities in saline. It was found that substantially all of the LPS in the NRS-formed precipitate dissolved leaving only about 1 percent of the recovered, precipitated LPS undissolved, while most of the LPS that precipitated from APRS did not dissolve in saline.
Dissolution of the saline-insoluble precipitates followed by SDS-PAGE analysis indicated that the APRS-formed precipitated complex contained a newly identified protein having an apparent relative molecular weight of about 60,000. That new protein was found to be a glycoprotein by staining with periodic acid-Schiff stain, and was referred to as gp60. Tobias and Ulevitch (1983) J. Immunol. 131:1913.
Data for the time-dependent shift of density and LPS precipitability from NRS and APRS showed similar time courses. In addition, SDS-PAGE analysis of precipitates taken at various times after admixture of LPS with APRS showed the gp60 material as well as possibly two other proteins of molecular weights of about 57,000 and about 79,000 interact with LPS to modify LPS/HDL binding kinetics. Tobias and Ulevitch (1983) J. Immunol. 131:1913.
It thus appeared that at least the before-described gp60 material was involved in mediating the binding of LPS to HDL. Subsequent work, discussed hereinafter, has however shown that that gp60 material is not the substance that retards binding between HDL and LPS, and it was believed that the homologous human protein was a known protein, an alpha.sub.2,beta.sub.1 -glycoprotein first reported by Iwasaki and Schmid (1970) J. Biol. Chem. 245:1814. That belief has now been found in error. The substance that retards binding is a newly identified glycoprotein, and is referred to hereinafter as lipopolysaccharide binding protein (LBP), or just a binding protein.